This invention relates generally to cell purification and, more specifically to methods for purifying cells based on the level of one or more products secreted by the cells.
The ability to obtain a purified cell line is fundamental in a growing number of basic research and applied commercial applications. For example, in drug discovery, use of a homogeneous population of cells that express a particular drug target allows for reproducible results, and therefore permits high-throughput screening. For this reason, a cell clone that stably expresses a drug target can be a prerequisite for initiating a drug screening campaign that can span several months and hundreds of thousands of candidate drugs.
Drug discovery efforts also depend on measuring cell responses, many of which can be in the form of secreting various cellular products that do not stay associated with the cell that produced them. For example, immune system cells can secrete numerous cytokines (interferons, interleukins and the like) that impact disease processes. The ability to easily purify cells based on their secretion response profile is therefore important. The absence of secretion of a particular product can be equally important in this setting.
In biopharmaceutical manufacturing, over $30 billion worth of products are produced annually, many from large-scale cultures of cloned cell lines producing a secreted protein. These products include monoclonal antibodies (for example, Herceptin® (anti-EGFR), Rituxan® (anti-CD20), Xolair® (anti-IgE)), cytokines (for example, Aranesp® (erythropoietin), Rebif® (interferon)), and numerous other proteins (for example, Factor VIII, TPA, FSH, BMP). Generation of cell lines for manufacturing these products is subject to many stringent requirements, including high protein secretion, low biomass production, adaptation to defined serum-free medium, and adaptation to bioreactor conditions. Isolation of a purified cell to generate a cell line can therefore be a critical aspect of preparing cell-based products. In the manufacturing setting, validation of cell cloning for product-producing cell lines is an added requirement. For example, one requirement of the United States Food and Drug Administration (FDA) is verification of the origin of each cell clone developed for manufacturing.
Currently, a variety of methods are used for purifying cells, such as to obtain a single cell for generating a clonal cell line. One technique involves seeding cells at low density, identifying cells/colonies with desirable attributes and isolating or collecting them by use of cloning rings or micropipette transfer. This approach provides visual verification of clonality at the time the cells/colonies are isolated. The considerable drawback is the slow and laborious procedure required to isolate and transfer each cell/colony into a new culture for evaluation. Further, this technique can be difficult to impossible to implement with cells that exhibit a low cloning efficiency, such as primary cells.
Another commonly used technique for purifying cells involves seeding cells at limiting dilution in multi-well plates (that is, maximizing the probability that many wells will receive only one cell). Under the best circumstances (for example, no cell clumping), one can expect ˜37% of wells to initially receive one cell. However, not all wells will result in cell growth, and wells receiving more than one cell usually have a growth advantage due to medium conditioning effects. Consequently, many of the “clones” generated from limiting dilution are not clonal, and three to five serial sub-cloning steps are required to improve the likelihood of achieving a clonal population. The success of limiting dilution can be improved by visual identification of wells receiving single cells, but this process is slow and laborious. Further, limiting dilution is difficult to implement with cells that have low cloning efficiency, such as primary cells. Finally, before a secreted product from a cell can be measured, the cell must be allowed to proliferate to obtain enough secreted product to be detected (such as by ELISA of the culture supernatant).
An additional commonly used technique for purifying cells involves flow cytometry. Flow cytometers process cells by suspending them in a fast-moving fluid stream, passing them through a laser beam/detector system to assess each cell's fluorescence and laser scattering characteristics, and then ejecting them from a nozzle within electrically-charged liquid droplets that are then deflected into a tube for collection. Although flow cytometry works well for non-adherent cell types (for example, blood cells), it is poorly suited for many other cell types (for example, neurons, hepatocytes) due to the harsh flow cytometry conditions, particularly when such cells are being sorted at one per well for cloning. Unfortunately, flow cytometry cannot be used to detect secreted cell products because the cells are suspended in a dynamic liquid stream. Although a bead-encapsulation method that allows secreted product detection by flow cytometry has been developed, this approach adds the complexities of encapsulating the cells, verifying the contents (that is, clonality) of each capsule, and then recovering the single cells of interest from the capsules.
Cell purification also is commonly accomplished by growing transfected cells in selective media. In selecting transfected mammalian cells, drug resistance is often used as a selection criterion because successfully transfected cells express both a protein of interest and a drug resistance gene product. Disadvantages of this approach include unintended physiological effects of the drug and the resistance gene product, and the current lack of acceptance by the FDA of this approach for production of biopharmaceuticals.
Thus, there exists a need for efficient methods for purifying cells based on their product-secretion profile. The invention satisfies this need and provides related advantages as well.